PROJECT SUMMARY/ABSTRACT Humans have more than 800 different G protein-coupled receptors (GPCRs), which form the largest family of cell-surface receptors and account for ~30% of all therapeutic drug targets. Upon activation by agonists, GPCRs adopt distinct activated states that allow them to couple either to G proteins, to initiate G protein-mediated signaling, or to GPCR kinases (GRKs), which phosphorylate GPCRs to enable their interactions with arrestins to terminate G protein activation and initiate arrestin-mediated signaling. While physiological agonists initiate both G protein- and arrestin-mediated signaling, often only one of the two pathways is therapeutically beneficial, while the other pathway is responsible for unwanted side effects. Given the importance of GPCRs as drug targets, there is huge interest in developing ?biased? GPCR agonist that signal predominantly through only one of the two pathways. However, the critical barrier for rationally developing biased agonist is the limited information on how GRKs interact with GPCRs and the complete absence of any high resolution GPCR/GRK structure. Crystal and cryo-EM structures of four GPCRs in complex with G proteins and of one GPCR in complex with an arrestin have provided an emerging understanding of the receptor conformations required for G protein and arrestin coupling, yet a mechanistic understanding of how GRKs bind and phosphorylate GPCRs have remained elusive due to the highly transient nature of this interaction. The objective of this proposal is to bridge this critical gap in knowledge by exploiting the interaction between rhodopsin and GRK1 as a paradigm to determine the mechanistic and structural basis of the targetable interaction between GPCRS and GRKs. SIGNIFICANCE: Completion of the proposed aims will reveal the first mechanistic basis of GRK/GPCR recognition and reveal the GPCR/GRK conformation that enables GRK binding and receptor phosphorylation. This information is of paramount importance for the rational design of therapeutic biased agonists for the treatment of a broad spectrum of diseases.